Microscopic pathology defines many human diseases, but the chemical composition of most histologically observed subcellular structures remains largely uncharacterized. Currently, there is no method for isolating subcellular areas of interest without disrupting the cells and thus distorting naturally occurring protein interactions. We combined in situ labeling schemes and subsequent microdissection with mass spectrometry to directly analyze subcellular complexes and organelles. To this end, we improved expression microdissection (xMD) in order to isolate optically dark targets (chemical, antibody, or metal-based stains) using optimized protocols compatible with mass spectrometric analysis to allow unsupervised thermoplastic capture of subcellular structures. In contrast to Laser Capture Microdissection, the film in xMD does not contain a dye and consequently the heat required for thermoplastic melting and adhesion is derived from the stained organelle. xMD is compatible with microscopic methodologies and avoids tissue homogenization. Combining the xMD technique with subsequent liquid chromatography coupled nano-spray mass spectrometry (LC-MS/MS) provides a novel and unique analytical method to identify the composition of subcellular components and characterize their physiological functions in cells. We introduced a custom-designed flashcube system that permits consistent and reproducible microdissection of nuclei across an FFPE rat brain tissue section in milliseconds. Both light and scanning electron microscopy demonstrated captured nuclear structures. Shotgun proteomic analysis of the samples showed a significant enrichment in nuclear localized proteins, with an average 25% of recovered proteins localized to the nucleus, versus 15% for whole tissue controls (p< 0.001). Targeted mass spectrometry using multiple reaction monitoring (MRM) showed more impressive data, with a 3-fold enrichment in histones, and a concurrentdepletion of proteins localized to the cytoplasm, cytoskeleton, and mitochondria. Our data demonstrate that the flashcube-xMD technology is applicable to the proteomic study of a broad range of targets in molecular pathology